The prior art and effectiveness of the present invention will both be illustrated using, as an example, the immunofluorescence testing of frozen sections.
In many patients, indirect immunofluorescence testing of frozen sections makes it possible to detect antibodies against the body's own tissue. The method was introduced by Coons et al (Coons A. H., Creech H. J., Jones R.N., Proc.Soc.Exp. Biol. N.Y., 47, 1941, p. 200ff).
The method, with reference to FIG. 1, may be explained as follows. In a first stage, a frozen section of healthy tissue is placed on a glass surface and is allowed to thaw and dry. As illustrated in FIGS. 1A-1C, it is covered with the dilute serum of a patient. If the serum contains antibodies against the antigens of the tissue, they remain attached to the frozen section. Antibodies which are not directed against antigens of the frozen section do not become attached and are washed off.
In a second stage, as illustrated in FIGS. 1D and 1E, antibodies obtained from animals and labelled with a fluorescent substance are then applied to the frozen section, these second stage antibodies being directed against the (already-attached) human antibodies (fluorescentlabelled antihuman serum). The (second stage) antibodies become attached to the (first stage) antihuman antibodies fixed to the frozen section and cannot be washed off.
Accordingly, if the patient's own serum contains antibodies against the tested tissue, the fluorescence microscope is able to detect the fluorescent label bonded to the corresponding tissue structures.
Frequently, direct frozen section immunofluorescence is used to investigate the tissue of patients in order to establish whether antibodies have become attached to certain tissue structures in vivo. For this purpose, frozen sections of the tissue are made and, in a direct immunofluorescence test, are brought directly together with a fluorescence-labelled antihuman serum, the first stage of the aforementioned indirect immunofluorescence test being omitted. The antibody-containing structures fluoresce in the washed products.
Different frozen section immunofluorescence techniques for physically manipulating frozen sections are known to those skilled in the art, but each has disadvantages. The techniques and their attendant disadvantages will be reviewed as part of the background discussion which follows.
It is standard practice when using immunofluorescence tests to mount the frozen sections on standard glass slides, a single slide generally being used for each frozen section. A detailed description of the test appears e.g. in Storch (Storch, W: "Immunfluoreszenzfibel", Fischer-Verlag, Jena, 1979). However, a number of sources of error are inherent in the test, which consequently requires considerable skill and large expenditures on labor and material.
A first source of error often occurs if the drops of sera added to the frozen sections run and the frozen sections then dry out. If the frozen sections become dry during the test, the results can generally not be utilized. Thus, as a precaution, large drops are prepared and reagents are wasted.
A second source of error can occur when preparing the section for microscopy. Before carrying out microscopy, the frozen section is covered with glycerin containing phosphate buffered saline and a cover slide is placed over it and should float on the glycerin. If there is an excessive dropwise addition of glycerin, however, the cover slide generally slips with the result that the microscope is contaminated and excess glycerin must be wiped away. However, removing glycerin may exacerbate the situation if too much glycerin is removed so that, as a result of capillary forces, the cover slide is drawn firmly onto the slide and squeezes the frozen section. If the cover slide is accidentally moved very tightly against the standard slide, the frozen section will likely be destroyed.
The same type of error may occur if, during microscopy, the frozen section is to be brought into the focal plane of the objective and the cover slide is brought too close to the objective. As a result, a positive result can appear negative.
Sera from different patients can be simultaneously tested on one slide. This allows combining operating sequences so that the testing process is simplified. Several frozen sections must be placed side-by-side on the slide beforehand and it must be ensured that there is no intermixing between the sera. Such simultaneous testing is facilitated by subdividing the slide into "reaction fields" which are set off from one another by a water-repelling coating on the slide (O'Neill, P., Johnson, G.D; Ann. N.Y. Acad. Sci. 177, 446-452, 1971; U.S. Pat. No. 3,736,042; EP-OS No. 79 103 987.8) or by color rings (Raisanen, S et al, J. Clin. Pathol. 33, 95-96, 1980). The more sera that are to be investigated on a slide, the smaller the preparatory expenditure during each individual test.
However, the aforementioned "simultaneous" testing technique is not conductive to carrying out more than about 20 individual tests on the same slide, particularly because the reaction fields are subject to successive dropwise application (of sera), resulting in different incubation times for the individual analyses. Testing more than about 20 samples thus generally produces too much variation in incubation time between the first and last sections. Additionally some sections might dry out and become unusuable during the period of time fluorescence-labelled antihuman serum is being added dropwise to other sections.
When large numbers of sera have to be tested side by side, e.g. 96 sera on one slide (see FIG. 2), the methods according to Stocker are available (EP-OS No. 79 103 987.8; DE-OS No. 3,107,964). Hydrophilic reaction fields are present in a congruent arrangement on two plates and are surrounded by a water-repelling coating. Frozen sections are placed on the reaction fields of one plate, while samples, e.g. serum dilutions or the fluorescence-labelled antihuman serum are added dropwise to the reaction fields of the other plate. Both plates are then placed in a frame in such a way that the frozen sections are immersed in the liquid samples. All the frozen sections of one plate are incubated for the same period of time and no frozen section dries out during the test, even during the application of the fluorescence-labelled antihuman serum.
The above method for testing large numbers of sera on one plate has hitherto been adopted in immunofluorescence diagnosis only in cases where it is possible to use suspendable antigens, because they can be added dropwise to the reaction fields, e.g. toxoplasmosis or loose exciters. However, those skilled in the technology of making frozen sections appreciate how difficult it can be using hitherto known processes to place 96 frozen sections cleanly and uniformly on the reaction fields of a plate. There is a high level of waste in industrial production.
It is often necessary to seek antibodies against various antigens in a serum. Several suspendable antigens can be added dropwise side by side on a reaction field and, after drying and optionally fixing, they are jointly covered with one serum dilution drop (Wang, S.P., Excerpta Medica, Amsterdam, 273-288, 1971). If one wishes to verify antibodies against different tissues, several frozen sections may be formed into a "composite section", which is then covered with a large sample or reagent drop. For this purpose, the frozen sections for each tissue can be individually produced and different frozen sections can be juxtaposed on one reaction field.
Alternatively, several fragments of different tissues can be jointly frozen into an aqueous solution of carboxymethylcellulose, followed by the simultaneous sectioning and mounting thereof (e.g. Nairn, R.C.: "Fluorescent Protein Tracing", Churchill Livingstone Edinburgh, 1976). Only a few organ fragments can be cut together in a block, however, and the fragments must be accurately trimmed to size. Moreover, this technique requires a great deal of skill and tissue may be lost. Also, frozen sections to be fixed in different ways cannot be juxtaposed in the same "composite section".
On one hand it is frequently the case that there is only little fluorescence-labelled antihuman serum or serum to be tested available for use. The test batch must then be kept as small as possible, the prerequisite for this being small frozen sections. The tissue is cut to the desired size and account is taken of the amount of material lost.
On the other hand, in the case of testing structures distributed in a non-uniform manner in the tissue, e.g. Langerhans islands of the pancreas or glomeruli of the kidneys, large sections are required in order to be sure that the desired structures are present during microscopy. It is otherwise extremely difficult (and perhaps prohibitively so) to cut islands or glomeruli from the loose frozen section.
It is frequently the case that the available organ fragments are so small that only very few frozen sections can be obtained therefrom, and the number of sections is not sufficient for the various tests to be performed. However, the structures which are of interest are frequently represented many times on these sections. An attempt can accordingly be made to subdivide the finished loose sections, either fresh or after freeze-drying. However, the desired structures are difficult to detect on the loose section and the manipulations involved in subdividing are generally very difficult.
A particular difficulty in biochemical tests on frozen sections is that the sections frequently adhere poorly to the substrate during incubation and often partly or wholly float off. If glass slides are used as the substrate, they must consequently be very carefully cleaned prior to the application of the frozen sections (chromosulphuric acid, ethanol, acetone; Storch W., see above). In certain laboratories, the glass surface is coated with glycerin and gelatin or with chicken protein (Romeis, B: "Mikroskopische Technik", Oldenbourg-Verlag, Munich, Vienna, 1968). This process constitutes no significant improvement, however, and is ignored by many scientists. For certain tissues, such as lungs, intestinal mucosa and particularly fat-rich tissue (e.g. pancreas, adrenal medulla), there has hitherto been no reliable process for maintaining unfixed frozen sections firmly on their substrate, particularly when long incubations are employed as part of the test procedure and where thorough washing must take place.
Consequently, it is therefore conventional practice to use unfixed tissue in immunohistochemistry (Wick, G. et al: "Immunofluorescence", Medizinische Verlagsgesellschaft Marburg/Lahn, 1978). Only if the antigen is soluble in water is the frozen section fixed, e.g. as in the case of the thyroid gland which has a colloid that may be made insoluble by treatment with absolute methanol.
Numerous processes are known enabling organic material to be bonded to activated surfaces. Particular efforts are being made to immobilize enzymes, antigens and antibodies on solids (e.g. Ternynck, T., Avrameas, S., FEBS-Letters 23, 24-28, 1972; Guesdon, J. L. et al, J. Immunol. Meth. 21,59-63, 1978; DE-AS No. 2,102,514; DE-OS No. 2,740,008; DE-OS No. 2,749,317; German Pat. No. 2,905,657). A description has already been given of cutting tissue embedded in polyacrylamide and bonding the sections chemically on a surface provided with reactive groups (Hausen, P., Dreyer, C., Stain Technol, 56, 287-293, 1981). It is therein assumed that it is the polyacrylamide which binds to the surface, not the tissue. However, the fact that a frozen section can firmly join itself (as unembedded tissue) to a surface which has been activated by being chemically treated so that it contains reactive groups, which section would not otherwise adequately adhere to an untreated surface, was clearly not recognized by these authors, and the technical literature otherwise fails to report thereon.